Expressions of P-selectin- and HSP72-like immunoreactivities in rat brain after transient middle cerebral artery occlusion

Abstract

The role of an adhesion molecule such as P-selectin may be important in the pathogenesis of stroke. However, temporal, spatial and cellular profiles of the expression of such a protein have not been fully studied. Change of immunoreactive P-selectin was examined in rat brain after transient middle cerebral artery (MCA) occlusion in comparison with that of 72 kDa heat shock protein (HSP72) which is a well known marker of cell injury. Western blot analyses were performed to ensure the selective detection of immunoreactive P-selectin and HSP72 proteins with each antibody using brain samples before and after ischemia. Temporal, spatial and cellular changes of immunohistochemical expressions of P-selectin and HSP72 were evaluated with rat brain sections at 2 and 8 h, and 1, 3 and 7 days of reperfusion after 1 h of MCA occlusion (MCAO). Hematoxylin-eosin (HE) staining was performed to evaluate brain cell damage at 3 and 7 days of reperfusion. Western blot showed a single band at molecular weights of 140 and 72 kDa for P-selectin and HSP72, respectively, only after ischemia. No significant band was observed without primary antibody. P-selectin-like immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 8 h to 1 day. The expression was diminished by 3 days of reperfusion. An immunoreactive HSP72 was scarcely present in the cerebral cortex and caudate of the sham control brain. However, the protein was induced in neurons of the MCA territory. The HSP72 induction was gradually intensified from 8 h with peaks at 1 day in the cortex and at 3 days in the caudate. The immunoreactivity decreased by 7 days. Histopathological study with HE staining showed no evident cell damage at 3 and 7 days of reperfusion. The present results indicate that temporal, spatial and cellular differences were present in the expressions of immunoreactive P-selectin and HSP72 proteins. P-selectin was expressed from an earlier stage of reperfusion in post-capillary venules, and the expression became maximum at the same time both in the cerebral cortex and caudate. In contrast, HSP72 induction began later in neurons and reached maximum at a different time between the cortex and caudate.